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1、BRITISH STANDARD BS EN ISO 11348-2:1999 Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 2: Method using liquid-dried bacteria The European Standard EN ISO 11348-2:1998 has the status of a British Standard
2、 ICS 13.060.70 BS EN ISO 11348-2:1999 This British Standard, having been prepared under the direction of the Health and Environment Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 March 1999 BSI 05-1999 ISBN 0 580 32235 1 National foreword T
3、his British Standard is the English language version of EN ISO 11348-2:1998. It is identical with ISO 11348-2:1998. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the responsibility to: aid enqui
4、rers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations re
5、presented on this subcommittee can be obtained on request to its secretary. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normally include an annex which lists normative references to international publications with their corresponding European publications. The Brit
6、ish Standards which implement these international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A
7、British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a
8、front cover, an inside front cover, pages i and ii, the EN ISO title page, page 2, the ISO title page, pages ii to iv, pages 1 to 11 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the in
9、side front cover. Amendments issued since publication Amd. No.DateComments BS EN ISO 11348-2:1999 BSI 05-1999i Contents Page National forewordInside front cover Foreword2 Forewordiii Text of ISO 11348-21 ii blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11348-2 December 1998 ICS 13.0
10、60.10 Descriptors: See ISO document English version Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 2: Method using liquid-dried bacteria (ISO 11348-2:1998) Qualit de leau Dtermination de leffet inhibiteu
11、r des chantillons deau sur la luminescence de Vibrio fischeri (Essai de bactries luminescentes) Partie 2: Mthode utilisant des bactries dshydrates (ISO 11348-2:1998) Wasserbeschaffenheit Bestimmung der Hemmwirkung von Wasserproben auf die Lichtemission von Vibrio fischeri (Leuchtbakterientest) Teil
12、2: Verfahren mit flssig getrockneten Bakterien (ISO 11348-2:1998) This European Standard was approved by CEN on 15 December 1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standa
13、rd without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other lan
14、guage made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
15、 Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of
16、 exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11348-2:1998 E EN ISO 11348-2:1998 BSI 05-1999 2 Foreword The text of the International Standard ISO 11348-2:1998 has been prepared by Technical Committee ISO/TC 147 “Water quality” in collaborati
17、on with Technical Committee CEN/TC 230 “Water analysis”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 1999, and conflicting national standards shal
18、l be withdrawn at the latest by June 1999. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland,
19、Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 11348-2:1998 was approved by CEN as a European Standard without any modification. NOTENormative references to International Standards are
20、 listed in Annex ZA (normative). EN ISO 11348-2:1998 ii BSI 05-1999 Contents Page Forewordiii Introduction1 1Scope1 2Normative references1 3Principle1 4Interferences1 5Reagents and materials2 6Apparatus2 7Sampling and sample pretreatment2 8Procedure3 9Evaluation3 10Expression of results4 11Criteria
21、of validity6 12Precision6 13Test report6 Annex A (informative) Colour-correction method7 Annex B (informative) Dilution level D Preparation of the dilution series9 Annex C (informative) Precision data10 Annex ZA (normative) Normative references to international publications with their relevant Europ
22、ean publications11 Table 1 Example of test evaluation Sample: effluent from a sewage treatment plant5 Table B.1 Preparation of the dilution series Composition of test and control batches10 Table C.1 Precision data for liquid-dried bacteria11 Descriptors: Water, quality, water pollution, water tests,
23、 bioassay, bacteria, light emission. EN ISO 11348-2:1998 BSI 05-1999iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO tech
24、nical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
25、 with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 %
26、of the member bodies casting a vote. International Standard ISO 11348-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. ISO 11348 consists of the following parts, under the general title Water quality Determination of the inhibitory effect of wat
27、er samples on the light emission of Vibrio fischeri (Luminescent bacteria test): Part 1: Method using freshly prepared bacteria; Part 2: Method using liquid-dried bacteria; Part 3: Method using freeze-dried bacteria. Annex A, Annex B and Annex C of this part of ISO 11348 are for information only. iv
28、 blank EN ISO 11348-2:1998 BSI 05-19991 Introduction Measurements according to this International Standard can be carried out using freshly prepared bacteria, as well as freeze-dried or liquid-dried bacterial preparations. Standardized work carried out by DIN NAW WI and ISO/TC 147/SC 5 WG 1 has show
29、n that in special cases these different techniques may give different results, especially where water samples contain heavy metals. Such varying sensitivity is caused by differences in media composition used in the preparation of freeze-dried or liquid-dried bacteria. These protective media influenc
30、e the bioavailability of toxicants and/or the light emission of luminescent bacteria. This means that the origin and type of preparation need to be taken into account when interpreting the results. This can be difficult sometimes, as freeze-dried and liquid-dried bacteria may be obtained from differ
31、ent suppliers. This in turn can mean that the composition is not known in detail or cannot be revised by the user. That is why in this International Standard additionally to toxicity measurements with liquid-dried bacteria (ISO 11348-2) and freeze-dried bacteria (ISO 11348-3) a procedure with freshl
32、y prepared bacteria is described (ISO 11348-1), the performance of which can be revised by the user in every detail. The laboratories responsible for the results have the opportunity to select the most suitable technique based on expert judgement and information about the water sample to be tested.
33、1 Scope ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B-11177). This part of ISO 11348 specifies a method using liquid-dried bacteria. This method is applicable to: waste water, aqueous extracts and leachate
34、s, fresh waters (surface or ground waters) or salt and brackish waters, especially the monitoring of changes in inhibition towards bacteria, pore water. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO
35、11348. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part of ISO 11348 are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC a
36、nd ISO maintain registers of currently valid International Standards. ISO 5667-16:1998, Water quality Guidance on biotesting of samples. ISO 7027:, Water quality Determination of turbidity1). 3 Principle The inhibition of light emission by cultures of Vibrio fischeri is determined by means of a batc
37、h test. This is accomplished by combining specified volumes of the test sample or the diluted sample with the luminescent bacteria suspension in a cuvette. The test criterion is the decrease of the luminescence, measured after a contact of 15 min and 30 min or optionally 5 min, taking into account a
38、 correction factor (fkt), which is a measure of intensity changes of control samples during the exposure time. The inhibitory effect of the water sample can be determined as LID (see Annex B) or as EC20 and/or EC50 values by means of a dilution series. The dilution level resulting in 0,5 mg/l is req
39、uired for the bioluminescence, samples with a high oxygen demand (and/or a low oxygen concentration) may cause a deficiency of oxygen and be inhibitory. An organic contamination of the sample by readily biodegradable nutrients (e.g. urea, peptone, yeast extract, usually W 100 mg/l) may cause a pollu
40、tant-independent reduction in bioluminescence. Salt concentrations in the initial sample exceeding 30 g/l NaCl or contents of other compounds giving equal osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. If the sample contains between 20 g/l and 50 g
41、/l NaCl-equivalents, no salt shall be added. The resulting concentration in the test samples shall not exceed the osmolarity of a 35 g/l sodium chloride solution. 5 Reagents and materials Chemicals of recognized analytical grade quality shall be used. Water shall be distilled or of equivalent purity
42、. 5.1 Test bacteria Strain of luminescent bacteria belonging to the species Vibrio fischeri NRRL B-11177. The bacterial suspensions used for toxicity measurements are prepared from commercially available liquid-dried reagents which can be stored in a freezer at 18 C to 20 C. The bacteria start glowi
43、ng immediately after reconstitution and are ready to be used for the test. 5.2 Sodium chloride solution, as diluent Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 litre with water. 5.3 Sodium hydroxide solution, c(NaOH) = 1 mol/l 5.4 Hydrochloric acid, c(HCl) = 1 mol/l NOTEFor the
44、 adjustment of the pH it may be necessary to use acids or bases of lower or higher concentration. 5.5 Solution for liquid-dried bacteria Dissolve in water, stir for about 30 min and adjust the pH to 7,0 0,2 with sodium hydroxide solution (5.3) or hydrochloric acid (5.4). Make up to 1 litre with wate
45、r. This solution can be stored in portions at 20 C. 5.6 Reference substances Zinc sulfate heptahydrate (ZnSO47H2O) 3,5-Dichlorophenol (C6H4OCl2) Potassium dichromate (K2Cr2O7) 6 Apparatus 6.1 Freezer for the storage of preserved bacteria. 6.2 Incubator or refrigerator to maintain the stock suspensio
46、n at a temperature of 3 C 3 C. 6.3 Thermostatically controlled thermoblock to maintain the test samples at a temperature of 15 C 1 C. Within one test the temperature deviation shall be at most 0,2 C. 6.4 Luminometer, measuring cell maintained at 15 C 1 C, equipped with suitable cuvettes. 6.5 Test tu
47、bes (vials), made of a chemically inert material, appropriate for the selected luminometer and having a capacity which facilitates the taking of a reading over the largest possible surface area. 6.6 pH-meter 6.7 Chronometer 6.8 Piston pipettes for plastic syringes, nominal capacity 10 4l, 500 4l and
48、 1 000 4l. 6.9 Piston pipettes with variable volume, 10 ml to 200 ml and 200 4l to 5 000 4l. 6.10 Refrigerated centrifuge 6.11 Water bath capable of maintaining a temperature of 20 C 2 C. 6.12 Conductometer 7 Sampling and sample pretreatment 7.1 Sampling Sampling shall be conducted in chemically ine
49、rt, clean containers in accordance with ISO 5667-16. Fill the containers completely and seal them. Test the samples as soon as possible after collection. Where necessary, store samples at a temperature of 2 C to 5 C in the dark in glass for not longer than 48 h. For periods up to two weeks, store at 20 C. Do not use chemicals to preserve the samples. Perform the necessary pH adjustment and salt addition just before testing. 8,0 gD(+)-Glucose monohydrate (C6H12O6H2